The current invention relates to the design, synthesis, and biochemical evaluation of chromogenic substrate compounds for sialidases of bacterial, viral, protozoa, and vertebrate (including human) origin. In particular, this invention provides a novel class of effective compounds as chromogenic substrates of these sialidases which yield chromogenic products after reactions catalyzed by sialidase take place. Also provided are methods of making these substrate compounds, methods of diagnosis and prognosis of sialidase related diseases using these substrate compounds.
Sialidase (EC 3.2.1.18, also known as neuraminidase, acylneuraminyl hydrolase) is a protein enzyme produced by many organisms such as bacteria, viruses, protozoa, and vertebrates including humans (Hirst, 1941). This class of enzymes catalyzes the hydrolysis of a terminal sialic acids which are xcex1-ketosidically linked to glycoproteins, glycolipids, and polysaccharides through an O-glycosidic bond (Drzeniek, 1972).
There are a large number of biological functions ascribed to sialidase enzyme, including cell-cell recognition phenomena and the pathogenicity of some infections by sialidase-bearing microorganisms (Schauer, 1985). In bacteria, sialidase helps bacterial adhesion to tissues, and provides additional nutritional sources (Crennell, et al., 1994). In the case of the influenza virus, sialidase is one of two surface glycoproteins and is considered to be important for both transporting the virus through mucin (Kienk and Rott, 1988) and for the elution of virus progeny from infected cells (Palese, et al., 1974). In a parasite, Trypanosoma cruzi, a sialidase (also known as trans-sialidase) removes sialic acids from infected cells and decorates its own surface with these sialic acids. In humans, silaidases are involved in protein digestion, immune responses, and cell proliferation. Abnormal production of sialidases may lead to serious human diseases such as sialidosis or increased Pseudomonas aeruginosa infection in cystic fibrosis patients.
Since sialidases are associated with many diseases, a color-producing substrate of sialidase would be an excellent diagnostic or prognostic reagent for sialidase-related diseases. For instance, sialidase level is elevated in bacterial vaginosis (Briselden, et al., 1992). Measurement of sialidase level in the vaginal samples could be used to diagnose bacterial vaginosis. In periodontal disease caused by bacterial infection, it has been shown that the presence of sialidase increases the colonization of harmful bacteria (Liljemark, et al., 1989). In influenza virus, viral sialidases are elevated in the mouth of patients. Measurement of sialidase level in the throat swab samples could be used to diagnose influenza virus. The cell invasion form of T. cruzi, Trypomastigote, expresses high levels of trans-sialidase activity; therefore, measurement of trans-sialidase level could be used for diagnosis of T. cruzi infection and for monitoring disease progress (Cross and Tackle, 1993). In cystic fibrosis patients, Pseudomonas aeruginosa infection is one of the leading causes of death. Sialidase was shown to be involved in the disease progress (Cacalano, et al., 1992). Sialidase is also related to the regulation of cell proliferation (Bratosin, et al., 1995), the clearance of plasma proteins (Bonten, et al., 1996), and the catabolism of gangliosides and glycoproteins (Gornati, et al., 1997).
The structure of sialidase has been extensively studied using numerous antigenic variants from several crystallographic studies (Colman, 1989, Varghese, et al., 1983; Colman, et al., 1983; Varghese, et al. 1992; Taylor and Itzstein, 1994). A key feature arising from these studies has been the fact that despite up to 50% variation in the primary sequence of the enzyme, the active site residues are highly conserved in both influenza A and B virus strains.
N-Acetylneuraminic acid, the product resulting from sialidase-mediated hydrolysis of polysaccharides, glycoproteins, and glycolipids, is shown below with the numbering system used to denote the carbon atoms: 
4-position modified N-acetylneuraminic acid analogs (Neu5Ac) have previously been described (Turner, et al., 1997):
2-O-(4-methylumbelliferyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-cyanoumbelliferyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(2-nitrophenyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-nitrophenyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-resorufin)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(5-bromo-4-chloro-3-indolyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(5-bromo-3-indolyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-indolyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-[4-(4nitrophenylazo)phenyl]-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-([4-(4-nitrophenylazo)resocinyl]-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-methoxyphenyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-dimethylaminophenyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-chloro-1-naphthyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(6-bromo-2-naphthyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-methylumbelliferyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(2-nitrophenyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-nitrophenyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-cyanoumbelliferyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-resorufin)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(5-bromo-4-chloro-3-indolyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(5-bromo-3-indolyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-indolyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-[4-(4nitrophenylazo)phenyl]-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-[4-(4-nitrophenylazo)resocinyl]-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-methoxyphenyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-dimethylaminophenyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(6-bromo-2-naphthyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-chloro-1-naphthyl)-4-deoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-methylumbelliferyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(2-nitrophenyl)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-methylumbelliferyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(2-nitrophenyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-nitrophenyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-cyanoumbelliferyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-resorufin)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(5-bromo-4-chloro-3-indolyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(5-bromo-3-indolyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-indolyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-[4-(4-nitrophenylazo)phenyl]-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-[4-(4-nitropheynazo)resorcinyl]-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-methoxyphenyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-dimethylaminophenyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-chloro-1-naphthyl)-4-ethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-methylumbelliferyl)-4-fluoro-N-acetylneuraminic acid-alpha-ketoside,
2-O-(2-nitrophenyl)-4-fluoro-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-nitrophenyl)-4-fluoro-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-cyanoumbelliferyl)-4-fluoro-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-resorufin)-4-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(5-bromo-4-chloro-3 indolyl)-4-fluoro-N-acetylneuraminic acid-alpha-ketoside,
2-O-(5-bromo-3-indolyl)-4-fluoro-N-acetylneuraminic acid-alpha-ketoside,
2-O-[4-(4-nitrophenylazo)phenyl]-4-fluoro-N-acetylneuraminic acid-alpha-ketoside,
2-O-[4-(4-nitropheynazo)resorcinyl]4-fluoro-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-methoxyphenyl)-4-fluoro-N-acetylneuraminic acid acid-alpha-ketoside,
2-O-[3-(dimethylamino)phenyl]-4-fluoro-N-acetylneuraminic acid acid-alpha-ketoside,
2-O-(4-chloro-1-naphthyl)-4-fluoro-N-acetylneuraminic acid acid-alpha-ketoside, and
2-O-(6-bromo-2-naphthyl)-4-fluoro-N-acetylneuraminic acid acid-alpha-ketoside.
Also, 4,7-alkoxy modified N-acetylneuraminic acid (Neu5Ac) analogs with viral sialidase have been reported (Liav, et al., 1998):
2-O-(4-methylumbelliferyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(2-nitrophenyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(4-nitrophenyl)-4,7-methoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-cyanoumbelliferyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(3-resorufin)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-(5-bromo-4-chloro-3-indolyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside, 2-O-(5-bromo-3-indolyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside, 2-O-(3-indolyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside, 2-O-[4-(4-nitrophenylazo)phenyl]-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside,
2-O-[4-(4-nitrophenylazo)resorcinyl]-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside, 2-O-(3-methoxyphenyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside, 2-O-(3-dimethylaminophenyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside, 2-O-(6-bromo-2-naphthyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside, 2-O-(4-chloro-1-naphthyl)-4,7-dimethoxy-N-acetylneuraminic acid-alpha-ketoside, as well as the corresponding 4,7-diethoxy, 4,7-dipropyl, and 4,7-dibutyl derivatives.
The applicant is unaware of any prior reports on the reactivity of 7-, 8-, 9-, 4,8-, 4,9-, 4,7,8,9-, 4,7,9-, 4,8,9-, 7,8-, 7,9-, 7,8,9-, or 8,9-position modified Neu5Ac analogues with viral sialidase.
The applicant is unaware of any prior reports on the reactivity of modified Neu5Ac analogs analogous to those presented herein with bacterial, vertebrate, or protozoal sialidase.
The substrate compounds of the current invention produce a visible color change upon hydrolysis, which is highly advantageous in medical diagnostic applications.
In one embodiment, the current invention relates to the design and synthesis of novel chromogenic substrate compounds for sialidases. In another embodiment, the subject invention pertains to the use of the novel chromogenic substrates in assays for the detection of sialidases. The sialidases which are detected using the procedures and compounds of the subject invention are of bacterial, viral, protozoa, and vertebrate (including human) origin. In a specific embodiment, the subject invention provides a novel class of compounds which are useful as chromogenic substrates of sialidases.
In one embodiment, the present invention provides chromogenic sialidase substrate compounds, analogues, pharmaceutically acceptable salts, derivatives, and mixtures thereof having the following formula: 
wherein, R1, R2, R4, and R5 can each, independently, be selected from the group consisting of H, R11, OC(O)R11, NO2, NHC(O)R11, Cl, Br, I, F, CHO, C(O)R11, C(Nxe2x80x94OH)NH2, OPO3R10, OPO2(CH2)jCH3, CH2PO3R10, OSO3R10, OSO2(CH2)jCH3, CH2SO3R10, and CN, where j is an integer from 0 to 3; wherein R3=NO2, CHO, (CR12xe2x95x90CR12)kCN or (CR12xe2x95x90CR12)kNO2, where k is an integer from 1 to 3, or 
wherein, R6, R7, R8, and R9 can each, independently, be selected from the group consisting of H, N3, R11, NO2, NHC(xe2x95x90NH)N(R10)2, NHC(O)R11, C(O)R11, Cl, Br, I, F, SR10, and (CH2)xC(xe2x95x90NH)N(R10)2 where x is an integer from 0 to 3; wherein R10=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein R11=R10, OR10, or N(R10)2; wherein, R12=H or (CH2)n; where n is an integer from 0 to 3.
Also provided are chromogenic sialidase substrate compounds, analogues, pharmaceutically acceptable salts, derivatives, and mixtures thereof having the formula of General Structure I, wherein, R1, R3, and R5 can each, independently, be selected from the group consisting of H, R11, OC(O)R11, NO2, NHC(O)R11, Cl, Br, I, F, CHO, C(O)R11, C(Nxe2x80x94OH)NH2, OPO3R10, OPO2(CH2)jCH3, CH2PO3R10, OSO3R10, OSO(CH2)jCH3, CH2SO3R10, or CN, where j is an integer from 0 to 3; wherein R2 or R4=H, R11, OC(O)R11, NO2, NHC(O)R11, Cl, Br, I, F, CHO, C(O)R11, C(Nxe2x80x94OH)NH2, OPO3R10, OPO2(CH2)jCH3, CH2PO3R10, OSO3R10, OSO2(CH2)jCH3, CH2SO3R10, CN,(CR12xe2x95x90CR12)kCN, and (CR12xe2x95x90CR12)kNO2, where j is an integer from 0 to 3, and where k is an integer from 1 to 3, or 
wherein, R6, R7, R8, and R9 can each, independently, be selected from the group consisting of H, N3, R11, NO2, NHC(xe2x95x90NH)N(R10)2, NHC(O)R11, C(O)R11, Cl, Br, I, F, SR10, and (CH2)xC(xe2x95x90NH)N(R10)2 where x is an integer from 0 to 3; wherein R10=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein R11=R10, OR10, or N(R10)2; wherein, R12=H or (CH2)n; where n is an integer from 0 to 3.
Also provided are chromogenic sialidase substrate compounds, analogues, pharmaceutically acceptable salts, derivatives, and mixtures thereof having the formula of General Structure I, wherein, R1 and R5 are each, independently, selected from the group consisting of H, R11, OC(O)R11, NO2, NHC(O)R11, Cl, Br, I, F, CHO, C(O)R11, C(Nxe2x80x94OH)NH2, OPO3R10, OPO2(CH2)jCH3, CH2PO3R10, OSO3R10, OSO2(CH2)jCH3, CH2SO3R10, CN, where j is an integer from 0 to 3, (CR12xe2x95x90CR12)kCN and (CR12xe2x95x90CR12)kNO2, where k is an integer from 1 to 3; wherein, R2, R3, and R4 can each, independently, be selected from the group consisting of H, R11, OC(O)R11, NO2, NHC(O)R11, Cl, Br, I, F, CHO, C(O)R11, C(Nxe2x80x94OH)NH2, OPO3R10, OPO2(CH2)jCH3, CH2PO3R10, OSO3R10, OSO2(CH2)jCH3, CH2SO3R10, and CN, where j is an integer from 0 to 3; wherein, R6, R7, R8, and R9 are each, independently, selected from the group consisting of H, N3, R11, NO2, NHC(xe2x95x90NH)N(R10)2, NHC(O)R11, C(O)R11, Cl, Br, I, F, SR10, (CH2)xC(xe2x95x90NH)N(R10)2, where x is an integer from 0 to 3; wherein R10=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein R11=R10, OR10, or N(R10)2; wherein R12=H or (CH2)n; where n is an integer from 0 to 3.
Also provided are chromogenic sialidase substrate compounds, analogues, pharmaceutically acceptable salts, derivatives, and mixtures thereof having the following formula: 
wherein, R1, R2, R3, and R4 are each, independently, selected from the group consisting of H, R11, OC(O)R11, NO2, Cl, Br, I, F, CHO, C(O)R11, C(Nxe2x80x94OH)NH2, OPO3R10, OPO2(CH2)jCH3, CH2PO3R10, OSO3R10, OSO2(CH2)jCH3, CH2SO3R10, and CN, where j is an integer from 0 to 3; wherein, R5=H or (CH2)kCH3, where k is an integer from 0 to 4; wherein, R6, R7, R8, and R9 are each, independently, selected from the group consisting of H, N3, R11, NO2, NHC(xe2x95x90NH)N(R10)2, NHC(O)R11, C(O)R11, Cl, Br, I, F, SR10, and (CH2)xC(xe2x95x90NH)N(R10)2, where x is an integer from 0 to 3; wherein, R10=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R11=R10, OR10, or N(R10)2.
Also provided are chromogenic sialidase substrate compounds, analogues, pharmaceutically acceptable salts, derivatives, and mixtures thereof having the following formula: 
wherein, R1=H, R8, OC(O)R8, NO2, NHC(O)R8, Cl, Br, I, F, CHO, C(O)R8, C(Nxe2x80x94OH)NH2, OPO3R7, OPO2 (CH2)jCH3, CH2PO3R7, OSO3R7, OSO2SO3R7, or CN, where j is an integer from 0 to 3; wherein, R2=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein R3, R4, R5, and R6 are each, independently, selected from the group consisting of H, N3, R8, NO2, NHC(xe2x95x90NH)N(R7)2, NHC(O)R8, C(O)R8, Cl, Br, I, F, SR7, and (CH2)xC(xe2x95x90NH)N(R7)2, where x is an integer from 0 to 3; wherein R7=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an in from 0 to 3; wherein, R8=R7, OR7, or N(R7)2.
Also provided are chromogenic sialidase substrate compounds, analogues, pharmaceutically acceptable salts, derivatives, and mixtures thereof having the following formula: 
wherein, R1=H, R8, OC(O)R8, NO2, Cl, Br, I, F, CHO, C(O)R8, C(Nxe2x80x94OH)NH2, OPO3R7, OPO2(CH2)jCH3, CH2PO3R7, OSO3R7, OSO2(CH2)jCH3, CH2SO3R7, or CN, where j is an integer from 0 to 3; wherein, R2=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R3, R4, R5, and R6 are each, independently, selected from the group consisting of H, N3, R8, NO2, NHC(xe2x95x90NH)N(R7)2, NHC(O)R8, C(O)R8, Cl, Br, I, F, SR7, and (CH2)xC(xe2x95x90NH)N(R7)2, where x is an integer from 0 to 3; wherein R7=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R8=R7, OR7, or N(R7)2.
Also provided are chromogenic sialidase substrate compounds, analogues, pharmaceutically acceptable salts, derivatives, and mixtures thereof having the following formula: 
wherein, R1, R2, R3, R4, R5, R6, and R7 are each, independently, selected from the group consisting of H, R13, OC(O)R13, NO2, Cl, Br, I, F, CHO, C(O)R13, C(Nxe2x80x94OH)NH2, OPO3R12, OPO2(CH2)jCH3, CH2PO3R12, OSO3R12, OSO2(CH2)jCH3, CH2SO3R12, and where j is an integer from 0 to 3; wherein, R8, R9, R10, R11 are each, independently, selected from the group consisting of H, N3, R13, NO2, NHC(xe2x95x90NH)N(R12)2, NHC(O)R13, C(O)R13, Cl, Br, I, F, SR12, and (CH2)xC(xe2x95x90NH)N(R12)2, where x is an integer from 0 to 3; wherein, R2=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer form 0 to 3; wherein, R13=R12, OR12, or N(R12)2.
Also provided are chromogenic sialidase substrate compounds, analogues, pharmaceutically acceptable salts, derivatives, and mixtures thereof having the following formula: 
wherein, R1, R2, R3, R4, R5, R6, and R7 are each, independently, selected from the group consisting of H, R 3, OC(O)R13, NO2, Cl, Br, I, F, CHO, C(O)R13, C(Nxe2x80x94OH)NH2, OPO3R12, OPO2(CH2)jCH3, CH2PO3R12, OSO3R12, OSO2(CH2)jCH3, CH2SO3R12, and CN, where j is an integer from 0 to 3; wherein, R8, R9, R10, and R11 are each, independently, selected from the group consisting of H, N3, R13, NO2, NHC(xe2x95x90NH)N(R12)2, NHC(O)R13, C(O)R13, Cl, Br, I, F, SR12, and (CH2)xC(xe2x95x90NH)N(R12)2, where x is an integer from 0 to 3; wherein, R12=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer form 0 to 3; wherein, R13=R12, OR12, or N(R12)2.
The subject invention further pertains to analogues, salts, derivatives, and mixtures of the exemplified compounds.